ProjectSummary Dendriticspinestabilityisdisruptedinpsychiatricandneurologicaldisorders.Dendriticspinesareenrichedina highlybranchedcytoskeleton,containingstableanddynamicfilamentous-(F-)actinpoolsandmonomericactin. LossoftheAbl2/Argnon-receptortyrosinekinase(Arg)anditsinteractionpartnercortactinfromdendriticspines causes widespread spine loss in late adolescence, but the mechanisms of how these two proteins support dendritic spine stability remain elusive. Our lab recently showed, using total internal reflection microscopy (TIRFM) single-filament based assays, that Arg and cortactin synergize to regulate Arp2/3-mediated actin branching. Coordinated regulation of the Arp2/3 complex via Arg and cortactin is a plausible mechanism by whichtheseproteinssupportspinestability.Inmyresearchplan,IwilltestthehypothesisthatArgandcortactin interact to confer dendritic spine stability via regulation of the Arp2/3 complex and maintenance of the spine stableactinpool. Aim1.TodeterminehowArginteractswithcortactinandtheArp2/3complextopromoteactinbranch nucleation. It remains unresolved how Arg regulates the Arp2/3 complex and how cortactin coordinates with Arg to enhance this effect. To address how Arg regulates the complex, I will learn how to conduct functional TIRFMsingle-filamentbasedassaystodefinetheminimaldomainofArgthatissufficienttoactivatetheArp2/3 complex.MypreliminarydataindicatethatArgcandirectlybindtheArp2/3complex.Iwillexpandthesebinding assaysandlearnhowtoconductchemicalcrosslinkingexperimentstodetermine(1)theminimalArgfragment thatcanmakeahighaffinityinteractionwiththeArp2/3complexand(2)thesubdomaincontactsthroughwhich ArginteractswiththeArp2/3complex,respectively.Finally,Iwilldeveloptwomethodstodeterminehowcortactin can coordinate with Arg to stimulate Arp2/3 activation. Two-color single molecule TIRF assays will address if cortactin recruits Arg to branch points and competition binding assays will determine if cortactin functions (mechanistically)toreleaseArgfromnascentbranchpoints,allowingfilamentelongation. Aim 2. To determine the mechanisms through which Arg and cortactin regulate spine stability. Arg, cortactin and the Arp2/3 complex are concentrated in dendritic spines and each is required for spine stability, buthowtheyinteracttoconferthisstabilityisnotunderstood.Preliminarydatacollectedinthelabsuggeststhat lossofcortactininitiallyreducesstableF-actinpriortodendriticspinedestabilization.Usingwell-definedArgor cortactinmutants,Iwillperformknockdown/complementationandconfocalmicroscopyinculturedhippocampal neuronstodeterminehowdisruptionsofArp2/3complexregulationaffectspinedynamicbehaviorandstability. IwilluseGFP-actinfluorescentrecoveryafterphotobleaching(FRAP),employingtheapproachesusedbymy collaborator,withthesamemutantcohorttodeterminehowtheseproteinsimpactspinestabilityviacontrolof thestableanddynamicactinpools.